Use of NRG4, or inhibitors thereof, in the treatment of colon and pancreatic cancers

ABSTRACT

This invention relates to compositions and uses of NRG4, and to variants thereof and to polynucleotides encoding NRG4, for therapeutic and diagnostic purposes, particularly related to colon and pancreatic cancer. This invention also relates to therapeutic agents based on or derived from the polynucleotides and proteins, NRG4 inhibitors, particularly antibodies capable of specifically binding to NRG4.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to nucleic acid sequences encoding amember of the neuregulin family, to polypeptides encoded by the nucleicacid sequences, inhibitors thereof, including antibodies and receptorsthat bind such polypeptides, and to their use in the treatment of colonand pancreatic cancer.

2. Description of the Related Art

Neuregulins (NRGs) belong to the epidermal growth factor (EGF)superfamily and are discussed in Carraway and Burden, Curr. Opin.Neurobiol. 5:1-7, 1995 and Yarden and Sliwkowski, Nature Rev. Mol. CellBiol. 2:127-137, 2001. There is a need in the art for agents and methodsuseful in modulating members of the NRG family. Due to their crucialroles in several essential cellular functions, including proliferationand differentiation, NRGs are important therapeutic targets for a numberof indications, including cancer, heart disease, wound healing,neurodegenerative diseases, and brain injury.

BRIEF SUMMARY OF THE INVENTION

The invention provides a composition comprising an isolated antibodythat binds specifically to an NRG4 polypeptide, wherein the isolatedantibody preferentially binds to NRG4 but not to other NRGs.

In preferred embodiments, the antibody is monoclonal, polyclonal or asingle-chain antibody.

Another object of the invention is to provide a method for detectingNRG4 polypeptides or mRNA for diagnostic and prognostic purposes.

In a preferred embodiment, the method utilizes an antibody which bindsan NRG4 polypeptide, and comprises contacting the antibody under bindingconditions with a biological sample from a human suspected of having anNRG4 protein-modulated disorder, and determining the amount of duplexformed between the antibody and the NRG4 protein in the biologicalsample, compared to a normal sample.

In another preferred embodiment, the method utilizes a polynucleotidethat binds to mRNA encoding an NRG4 polypeptide under stringentconditions, such method comprising contacting nucleic acid of thebiological sample with the polynucleotide under binding conditions toform a duplex, and determining the amount of the duplex formed, comparedto a normal sample.

Another object of the invention is to provide methods of modulating theamount of NRG4 protein activity in a subject, using NRG4 polypeptide,NRG4-encoding polynucleotide, polypeptides representing NRG4 antisense,and NRG4 antibody or receptor compositions.

In one embodiment, the method comprises administering an effectiveamount of a composition comprising an NRG4 polypeptide or an antibodythat binds to an NRG4 polypeptide.

In another embodiment, the method comprises administering an effectiveamount of a composition consisting of an NRG4-encoding polynucleotide,or a polynucleotide complementary to all or part of natural mRNAsencoding NRG4 polypeptides.

A further object of the invention is to provide methods for treating anNRG4 protein-modulated disorder in a subject, using an NRG4 polypeptide,NRG4-encoding polynucleotide, polynucleotides complementary to NRG4encoding polynucleotides, or NRG4 antibody compositions.

In one embodiment, the method comprises administering an effectiveamount of a composition comprising an NRG4 polypeptide or an antibodythat binds to an NRG4 polypeptide of the invention, wherein thecomposition further comprises a pharmaceutically acceptable carrier.

In another embodiment, the method comprises administering an effectiveamount of a composition consisting of an NRG4-encoding polynucleotide orpolynucleotide representing NRG4 antisense, a ribozyme, or RNAi, whereinthe composition further comprises a pharmaceutically acceptable carrier.

In a preferred embodiment, the method is accomplished by implantingcells containing a polynucleotide encoding an NRG4 polypeptide of theinvention into the patient, wherein the cells express NRG4 polypeptidein the patient, and wherein the implanted cells optionally contain aheterologous or recombinant DNA expression construct for expressing NRG4polypeptide.

In a most preferred embodiment, the implanted cells are encapsulated ina semipermeable membrane.

In another embodiment of the invention, a patient is treated with atherapeutically effective amount of a polynucleotide capable ofhybridizing to an NRG4-encoding polynucleotide or complement thereof.

In preferred embodiments, the polynucleotide is an antisense construct,a ribozyme, or RNAi.

In another preferred embodiment, the polynucleotide is a viral, such asa retroviral, vector comprising a promoter and an NRG4-encodingpolynucleotide or a complement thereof.

In yet another embodiment a patient is treated with a therapeuticallyeffective amount of a polypeptide capable of binding an NRG4polypeptide.

In a most preferred embodiment, the polypeptide is an antibody.

In another preferred embodiment, the polypeptide is a wild-type ormutant receptor for NRG4.

The invention further provides a method for diagnosing a NRG4protein-modulated disorder using a biological sample from a humansuspected of having the disorder, the method comprising:

-   -   (a) providing an antibody that binds to an NRG4 polypeptide;    -   (b) contacting the antibody with the sample under binding        conditions to form a duplex; and    -   (c) determining the amount of duplex formed, compared to a        normal sample.

The invention also provides a method for diagnosing a NRG4protein-modulated disorder in a biological sample from a human suspectedof having the disorder, the method comprising:

-   -   (a) providing a polynucleotide that binds to mRNA encoding an        NRG4 polypeptide under stringent conditions;    -   (b) contacting nucleic acid of the sample with the        polynucleotide under binding conditions to form a duplex; and    -   (c) determining the amount of the duplex formed, compared to a        normal sample.

The invention still further provides a method for modulating the amountof NRG4 protein activity in a subject, the method comprisingadministering an effective amount of a composition selected from a groupconsisting of:

-   -   (a) an NRG4 polypeptide;    -   (b) an antibody that binds to an NRG4 polypeptide; and    -   (c) a wild-type or mutant receptor for NRG4.

The invention further provides a method for modulating the amount of aNRG4 protein in a subject, comprising administering an effective amountof a composition comprising a polynucleotide encoding NRG4.

The invention further provides a method for treating an NRG4protein-modulated disorder in a subject, comprising administering to thesubject an effective amount of a composition selected from a groupconsisting of:

-   -   (a) an NRG4 polypeptide;    -   (b) an antibody that binds an NRG4 polypeptide; and    -   (c) a wild-type or mutant receptor for NRG4, wherein the        composition further comprises a pharmaceutically acceptable        carrier.

The invention also provides a method for treating a NRG4protein-modulated disorder in a subject, comprising administering to thesubject an effective amount of a composition consisting of an NRG4polynucleotide (sense or antisense), wherein the composition furthercomprises a pharmaceutically acceptable carrier.

The invention further provides a method of providing trophic support forcells in a patient in need thereof, the method comprising administeringto the patient a composition selected from the group consisting of apolynucleotide encoding an NRG4 polypeptide comprising SEQ ID NO:2, andan NRG4 polypeptide. The composition may additionally or alternativelycomprise fragments of NRG4 polypeptide, such as amino acids 1-45; 2-45,or 9-45.

In one embodiment, the polynucleotide is administered by implantingcells which express the polynucleotide into the patient, wherein thecells express NRG4 polypeptide in the patient, and in a specificembodiment the implanted cells are encapsulated in a semipermeablemembrane.

In a further embodiment of this method, the patient suffers from acondition selected from the group consisting of pancreatic cancer andcolon cancer.

In specific embodiments of this method, the polynucleotide is anantisense construct, ribozyme, RNAi, or a viral vector comprising apromoter.

The invention yet further provides a method of treating a patient withan NRG4 protein-modulated disorder, such as colon cancer, the methodcomprising administering a composition comprising a therapeuticallyeffective amount of polypeptides capable of binding NRG4 or a variantthereof.

In a specific embodiment of this method, the polypeptides areantibodies.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph showing expression of NRG4 in a variety of celllines, relative to actin expression.

FIG. 2 is a bar graph showing expression of NRG4 in human adult tissues,relative to GusB expression.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 is the polynucleotide sequence of NRG4.

SEQ ID NO:2 is the amino acid sequence of NRG4.

SEQ ID NO:3 is the forward primer used to amplify NRG4.

SEQ ID NO:4 is the reverse primer used to amplify NRG4.

SEQ ID NO:5 is the forward primer used to amplify actin.

SEQ ID NO:6 is the reverse primer used to amplify actin.

SEQ ID NO:7 is the forward primer used to amplify GusB.

SEQ ID NO:8 is the reverse primer used to amplify GusB.

DETAILED DESCRIPTION OF THE INVENTION

Neuregulins are a family of structurally related polypeptides, membersof which regulate cellular proliferation, differentiation, apoptosis,and cell survival (reviewed in Carraway and Burden, Curr. Opin.Neurobiol 5:1-7, 1995). In vivo studies demonstrated that targeteddisruption of the neuregulin-1 gene is associated with cardiac andneural abnormalities, indicating that neuregulins play an important rolein cardiac and neural development (Meyer and Birchmeier, Nature378:386-390, 1995). Furthermore, expression and signaling by certainneuregulin family members was altered following nerve injury anddegeneration (Carroll et al., J. Neurosc. 17:1642-1659,1997). Thus,neuregulins are important therapeutic targets for a number ofindications, including, for example, cancers such as breast cancer,prostate cancer, pancreatic cancer, colon cancer and ovarian cancer;heart diseases and injuries such as myocardial infarction and ischemia;wound healing such as bone fractures, tissue repair and regeneration;skin conditions such as burns, cuts, and ulcers; neurological conditionssuch as neuro-degenerative disease and stroke, multiple sclerosis,peripheral neuropathy, amyotrophic lateral sclerosis, dementia,Alzheimer's disease, Parkinson's disease, and Huntington's disease,brain injury, acute spinal cord injury, nervous system injury andperipheral nerve injury; infection, epilepsy, and acoustic trauma.

Neuregulins mediate cellular responses through interactions with theirreceptors, transmembrane tyrosine kinases of the ErbB family (reviewedin Burden and Yarden, Neuron 18:847-855, 1997). These receptors functionas homo- and heterodimers. Different receptor combinations exhibitdifferent binding affinities for neuregulins and different abilities toactivate downstream signaling complexes and promote cell proliferation.Neuregulin-1 (NRG1) and neuregulin-2 (NRG2) bind directly to the ErbB3and ErbB4 receptor but can recruit ErbB1 and ErbB2 as coreceptors.Targeted disruption in mice of the genes encoding ErbB2 and ErbB4results in heart malformations and defects in the nervous system (Lee etal., Nature 378:386-390, 1995; Gassmann et al., Nature 378:390-394,1995). However, the specific neural defects differ between ErbB2 andErbB4 homozygous mutant mice (Carraway, BioEssays 18:263-266, 1996).These findings demonstrate that specific receptors play different rolesin cell growth and development.

The neuregulins constitute a subfamily of the epidermal growth factor(EGF) family. Neuregulins contain a characteristic receptor-bindingEGF-like motif with six conserved cysteine residues. Neuregulinprecursors may contain other motifs, including a signal peptide, acysteine-containing N-terminal domain, either a cysteine-rich domain(CRD) or a domain distantly related to a kringle motif, an Ig-like loop,a glycosylation domain, and a transmembrane domain (Burden and Yarden,Neuron 18:847-855, 1997). Depending on the presence or absence of thetransmembrane domain, neuregulin precursors may be either transmembraneproteins or secreted polypeptides. The most highly conserved region ofneuregulins is their EGF-like domain.

Four members of the neuregulin family have been identified, includingNRG1, NRG2, NRG3, and NRG4. Multiple isoforms of NRG1 and NRG2 exist,some of which display heterogeneous binding affinities for differentErbB receptors (Tzahar et al., J. Biol. Chem. 269:25226-25233, 1994;Pinkas-Kramarski et al., Mol. Cell Biol. 18:6090-6101, 1998). Thedifferent NRG1 isoforms result from alternative splicing of a singlegene (Carraway, BioEssays 18:263-266,1996). The present inventionemploys NRG4 polypeptides and polynucleotides encoding NRG4. NRG4 isdisclosed in applicants' co-pending application PCT/US00/22326. NRG4 isexpressed in specific tissues during development and promotes meioticmaturation of Xenopus oocytes. In humans, NRG4 is expressed inparticular in skeletal muscle and pancreas cells, and NRG4 levels arealtered in pancreatic tumor tissue as compared to normal pancreaticsamples.

NRG4 Nucleic Acids, Polypeptides and Antibodies.

The nucleotide sequence encoding a cDNA copy of the coding region forNRG4 is shown in SEQ ID NO:1. The predicted polypeptide sequence forNRG4 is shown in SEQ ID NO:2. These sequences were first disclosed inapplicants' co-pending application PCT/US00/22326.

Polynucleotide molecules of the invention can be made using thetechniques of synthetic chemistry given the sequences disclosed herein.The degeneracy of the genetic code permits alternate nucleotidesequences to be synthesized which will encode the amino acid sequence ofSEQ ID NO:2. Use of all such nucleotide sequences is within the scope ofthe present invention.

The present invention employs nucleic acid sequences that encode NRG4.Also included within the scope of the methods of the invention aresequences that are substantially the same as the nucleic acid sequencesencoding NRG4. Such substantially same sequences may, for example, besubstituted with codons more readily expressed in a given host cell suchas E. coli according to well known and standard procedures. The presentinvention also employs nucleic acid sequences that will hybridize tosequences which encode NRG4 or complements thereof. Such nucleic acidsequences can be at least 90%, 91 %, 92%, 93% or 94% identical,preferably 95%, 96%, 97%, 98% or 99% identical. The invention employsnucleic acid sequences encoding functional domains of NRG4. In addition,the invention employs nucleic acids that encode polypeptides that arerecognized by antibodies that bind NRG4 polypeptides. Polynucleotidesencoding such polypeptides can range in size from 10, 25, 50, 75, 125,150, 175, 200, 225, 250, 275, 300, 325, 335, or 345 contiguousnucleotides of SEQ ID NO:1, although this list is not intended to belimiting and intermediate lengths may also be suitable.

The present invention employs vectors comprising expression regulatoryelements operably linked to any of the nucleic acid sequences employedwithin the scope of the invention. Vectors may include, but are notlimited to, plasmids, episomes, retroviruses, lentivirus, adenovirus,and parvoviruses including adeno-associated virus. This invention alsoemploys host cells, of any variety, that have been transformed orinfected or transfected with vectors comprising expression regulatoryelements operably linked to any of the nucleic acid sequences includedwithin the scope of the present invention. Examples of host cellsinclude bacterial cells, yeast cells, plant cells, tissue culture cellsincluding primary, immortalized and transformed cell lines, and insectcells.

Reference to NRG4 herein is intended to include growth factors of anyorigin which are substantially homologous to and which are biologicallyequivalent to the NRG4 characterized and described herein. Suchsubstantially homologous growth factors may be native to any tissue orspecies and, similarly, biological activity can be characterized in anyof a number of biological assay systems. For example, assays formeasuring NRG4 activity are disclosed in PCT/US00/22326 and Harari etal., Oncogene 18:2681-2689, 1999, both of which are incorporated byreference herein.

The term “biologically equivalent” is intended to mean that thecompositions of the present invention are capable of demonstrating someor all of the same growth properties in a similar fashion, notnecessarily to the same degree as the NRG4 as described herein orrecombinantly produced NRG4 of the invention.

The invention also employs fragments of NRG4. Preferred fragmentsinclude: amino acids from about residue 9 to about residue 45 as shownin SEQ ID NO:2; amino acids from about residue 4 to about residue 50;amino acids from about residue 4 to about residue 82; amino acids fromabout residue 1 or 2 to residue 50; amino acids about residue 1 or 2 toresidue 64; and amino acids from about residue 1 or 2 to about residue82 as shown in SEQ ID NO: 2. Such fragments can be prepared from theprotein by standard biochemical methods or by expressing apolynucleotide encoding the fragment.

Also employed within the scope of the invention are NRG4 molecules thatdiffer from native NRG4 by virtue of changes in biologically activesites. NRG4 has a putative transmembrane domain at amino acid residues64-82. An NRG4 molecule that does not include this transmembrane domaincan be prepared by expressing DNA encoding NRG4, wherein thecorresponding codons for some or all of amino acid residues 64-82 havebeen deleted. DNA encoding NRG4 with altered receptor binding canlikewise be produced. The EGF-like receptor binding domain of NRG4extends from approximately amino acid 4 to amino acid 50, moreparticularly from about amino acid 9 to about amino acid 45. Forexample, it may be desirable to alter receptor specificity of NRG4 bysubstituting the receptor binding regions of a different neuregulin orother EGF-like domain for that of NRG4.

Also included within the meaning of substantially homologous is any NRG4which may be isolated by virtue of cross-reactivity with antibodies tothe NRG4 described herein or whose encoding nucleotide sequencesincluding genomic DNA, mRNA or cDNA may be isolated throughhybridization with the complementary sequence of genomic or subgenomicnucleotide sequences or cDNA of the NRG4 herein or fragments thereof. Itwill also be appreciated by one skilled in the art that degenerate DNAsequences can encode NRG4 and these are also intended to be includedwithin the methods and compositions of the present invention, as areallelic variants of NRG4.

Recombinant NRG4 may be made by expressing the DNA sequences encodingNRG4 in a suitable transformed host cell. Using methods well known inthe art, the DNA encoding NRG4 may be incorporated into an expressionvector, transformed into a host cell and conditions established that aresuitable for expression of NRG4 by the transformed cell.

The DNA encoding NRG4 can be engineered to take advantage of preferredcodon usage of host cells. Codon usage in Pseudomonas aeruginosa isdescribed in, for example, West et al., Nucleic Acids Res. 11:9323-9335,1988. Codon usage in Saccharomyces cerevisiae is described in, forexample, Lloyd et al., Nucleic Acids Res. 20:5289-5295, 1992. Codonpreference in Corynebacteria and a comparison with Escherichia colipreference is provided in Malubres et al., Gene 134:15-24, 1993. Codonusage in Drosophila melanogasteris described in, for example, Akashi,Genetics 136:927-935, 1994.

Any suitable expression vector may be employed to produce recombinantNRG4. For example, insect cells such as Baculovirus can also be employedas expression systems. A preferred method is expression in insect cells,such as Tn5 or Sf9 cells, using baculovirus vector. Other methods caninclude E. coli, yeast cells, etc.

The invention employs polypeptide fragments of NRG4. Polypeptidefragments of the invention can comprise at least 8, 10, 12, 15, 18, 19,20, 25, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, or 112contiguous amino acids selected from SEQ ID NO:2.

Exemplary 20 amino acid fragments include the following based on SEQ IDNO:2: 1-20, 2-21, 3-22, 4-23, 5-24, 6-25, 7-26, 8-27, 9-28, 10-29,11-30, 12-31, 13-32, 14-33, 15-34, 16-35, 17-36, 18-37, 19-38, 20-39,21-40, 22-41, 23-42, 24-43, 25-44, 26-45, 27-46, 28-47, 29-48, 30-49,31-50, 32-51, 33-52, 34-53, 35-54, 36-55, 37-56, 38-57, 39-58, 40-59,41-60, 42-61, 43-62, 44-63, 45-64, 46-65, 47-66, 48-67, 49-68, 50-69,51-70, 52-71, 53-72, 54-73, 55-74, 56-75, 57-76, 58-77, 59-78, 60-79,61-80, 62-81, 63-82, 64-83, 65-84, 66-85, 67-86, 68-87, 69-88, 70-89,71-90, 72-91, 73-92, 74-93, 75-94, 76-95, 77-96, 78-97, 79-98, 80-99,81-100, 82-101, 83-102, 84-103, 85-104, 86-105, 87-106, 88-107, 89-108,90-109, 91-110, 92-111, 93-112, 94-113, 95-114, and 96-115.

Exemplary 25 amino acid fragments include the following based on SEQ IDNO:2: 1-25, 2-26, 3-27, 4-28, 5-29, 6-30, 7-31, 8-32, 9-33, 10-34,11-35, 12-36, 13-37, 14-38, 15-39, 16-40, 17-41, 18-42, 19-43, 20-44,21-45, 22-46, 23-47, 24-48, 25-49, 26-50, 27-51, 28-52, 29-53, 30-54,31-55, 32-56, 33-57, 34-58, 35-59, 36-60, 37-61, 38-62, 39-63, 40-64,41-65, 42-66, 43-67, 44-68, 45-69, 46-70, 47-71, 48-72, 49-73, 50-74,51-75, 52-76, 53-77, 54-78, 55-79, 56-80, 57-81, 58-82, 59-83, 60-84,61-85, 62-86, 63-87, 64-88, 65-89, 66-90, 67-91, 68-92, 69-93, 70-94,71-95, 72-96, 73-97, 74-98, 75-99, 76-100, 77-101, 78-102, 79-103,80-104, 81-105, 82-106, 83-107, 84-108, 85-109, 86-110, 87-111, 88-112,89-113, 90-114, and 91-115.

Biologically Active Variants

Variants of the protein and polypeptides disclosed herein can also beemployed in the invention. Variants can be naturally or non-naturallyoccurring. Naturally occurring variants are found in humans or otherspecies and comprise amino acid sequences which are substantiallyidentical to the amino acid sequence shown in SEQ ID NO:2. Specieshomologs of the protein can be obtained using subgenomic polynucleotidesof the invention or fragments thereof as suitable probes or primers toscreen cDNA libraries from other species, such as mice, monkeys, oryeast, identifying cDNAs which encode homologs of the protein, andexpressing the cDNAs as is known in the art.

Non-naturally occurring variants, which retain substantially the samebiological activities as naturally occurring protein variants, are alsoincluded here. Preferably, naturally or non-naturally occurring variantshave amino acid sequences which are at least 85%, 90%, or 95% identicalto the amino acid sequence shown in SEQ ID NO:2. Harari et al., Oncogene18:2681-2689 (1999), disclose methods for assaying NRG4 activity,including binding to ErbB proteins, signal activation, stimulation ofproliferation of ErbB-4-expressing cells, and recognition and activationof ErbB-4. Biological activities of EGFH2 proteins are also disclosed inPCT/US00/22326, including mitogenic effects.

More preferably, the molecules are at least 98% or 99% identical.Percent identity is determined using any method known in the art. Anon-limiting example is the Smith-Waterman homology search algorithmusing an affine gap search with a gap open penalty of 12 and a gapextension penalty of 1. The Smith-Waterman homology search algorithm istaught in Smith and Waterman, Adv. Appl. Math. 2:482-489, 1981.

Guidance in determining which amino acid residues can be substituted,inserted, or deleted without abolishing biological or immunologicalactivity can be found using computer programs well known in the art,such as DNASTAR software. Preferably, amino acid changes areconservative amino acid changes, i.e., substitutions of similarlycharged or uncharged amino acids. A conservative amino acid changeinvolves substitution between members of a family of amino acids whichare related in their side chains. Naturally occurring amino acids aregenerally divided into four families: acidic (aspartate, glutamate),basic (lysine, arginine, histidine), non-polar (alanine, valine,leucine, isoleucine, proline, glycine, phenylalanine, methionine,tryptophan), and uncharged polar (asparagine, glutamine, cysteine,serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, andtyrosine are sometimes classified jointly as aromatic amino acids.

It is reasonable to expect that an isolated replacement of a leucinewith an isoleucine or valine, an aspartate with a glutamate, a threoninewith a serine, or a similar replacement of an amino acid with astructurally related amino acid will not have a major effect on thebiological properties of the resulting variant. Guidance concerningwhich amino acid changes are likely to be phenotypically silent can befound in Bowie, J. U., et al., “Deciphering the Message in ProteinSequences: Tolerance to Amino Acid Substitutions,” Science247:1306-1310, 1990.

Variants of the NRG4 protein used herein include glycosylated forms,aggregative conjugates with other molecules, and covalent conjugateswith unrelated chemical moieties. Covalent variants can be prepared bylinking functionalities to groups which are found in the amino acidchain or at the N- or C-terminal residue, as is known in the art.Variants also include allelic variants, species variants, and muteins.Truncations or deletions of regions which do not affect functionalactivity of the proteins are also variants.

The invention further employs variations of the NRG4 polypeptide whichshow comparable expression patterns to those disclosed herein inrelation to pancreatic and colon cancer, or which include antigenicregions. Such mutants include deletions, insertions, inversions,repeats, and type substitutions.

Of particular interest are substitutions of basic or positively chargedamino acids with another charged amino acid and with neutral ornegatively charged amino acids. The latter results in proteins withreduced positive charge to improve the characteristics of the disclosedprotein. The prevention of aggregation is highly desirable. Aggregationof proteins not only results in a loss of activity but can also beproblematic when preparing pharmaceutical formulations, becauseaggregates can be immunogenic. (Pinckard et al., Clin. Exp. Immunol.2:331-340 1967; Robbins et al., Diabetes 36:838-845, 1987; Cleland etal., Crit. Rev. Therapeutic Drug Carrier Systems 10:307-377, 1993).

Amino acids in the NRG4 polypeptides employed in the present inventionthat are essential for function can be identified by methods known inthe art, such as site-directed mutagenesis or alanine-scanningmutagenesis (Cunningham and Wells, Science 244:1081-1085,1989). Thelatter procedure introduces single alanine mutations at many, if notevery residue position in the molecule. The resulting mutant moleculesare then tested for biological activity such as binding to a natural orsynthetic binding partner. Sites that are critical for ligand-receptorbinding can also be determined by structural analysis such ascrystallization, nuclear magnetic resonance or photoaffinity labeling(Smith et al., J. Mol. Biol. 224:899-904, 1992; and de Vos et al.Science 255:306-312, 1992).

Antibodies to NRG4

Antibodies to NRG4 are employed in the invention. Antibodies of theinvention can be used, for example, to detect NRG4 polypeptides oncells, in culture medium, or in biological samples, such as blood ortissue samples. Antibodies may be polyclonal, monoclonal or single chainantibodies. Antibodies to NRG4 protein or an epitope thereof can be madeby any of a number of methods known in the art. Detailed methods forgenerating antibodies are provided in Harlow and Lane, Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratories, 1988, which isincorporated by reference. Methods of generating the antibodies are alsodescribed in U.S. patent application Ser. No. 08/988,671, which isincorporated by reference in its entirety. By epitope, reference is madeto an antigenic determinant of a polypeptide. An epitope could compriseat least 6, 7, 8, 9, 10, or 11 amino acids in a spatial conformationwhich is unique to the epitope. Methods of determining the spatialconformation of amino acids are known in the art, and include, forexample, x-ray crystallography and 2 dimensional nuclear magneticresonance. Antibodies to NRG4 can also be raised against oligopeptidesthat include one or more conserved regions such that the antibody cancross-react with other species and/or family members.

Polyclonal antibodies can be prepared by immunizing rabbits or otheranimals by injecting antigen followed by subsequent boosts with antigenat appropriate intervals. Animal sera is assayed for immunoreactivityagainst NRG4 by any of a number of methods, including, for example,Western blot or ELISA. Monoclonal antibodies can be prepared after themethod of Milstein and Kohler by fusing splenocytes from immunized miceor other animals, such as rats or rabbits, with continuously replicatingtumor cells (Milstein and Kohler, Nature 256:495-497, 1975).

Techniques for purifying antibodies are those available in the art. In apreferred embodiment, antibodies are affinity purified by passing theantibodies over a column to which amino acid sequences of the inventionare bound. Bound antibody is then eluted. Any technique may be chosen topurify antibodies of the invention.

Methods well known in the art may be used to generate antibodies,polyclonal antisera, or Monoclonal antibodies that are specific for anNRG4 polypeptide. Antibodies also may be produced as geneticallyengineered immunoglobulins (Ig) or Ig fragments designed to havedesirable properties. For example, by way of illustration and notlimitation, antibodies may include a recombinant IgG that is a chimericfusion protein having at least one variable (V) region domain from afirst mammalian species and at least one constant region domain from asecond, distinct mammalian species. Most commonly, a chimeric antibodyhas murine variable region sequences and human constant regionsequences. Such a murine/human chimeric immunoglobulin may be“humanized” by grafting the complementarity determining regions (CDRs)derived from a murine antibody, which confer binding specificity for anantigen, into human-derived V region framework regions and human-derivedconstant regions. Fragments of these molecules may be generated byproteolytic digestion, or optionally, by proteolytic digestion followedby mild reducton of disulfide bonds and alkylation. Alternatively, suchfragments may also be generated by recombinant genetic engineeringtechniques.

As used herein, an antibody is said to be “immunospecific” or to“specifically bind” an NRG4 polypeptide if it reacts at a detectablelevel with the NRG4, preferably with an affinity constant, K_(a), ofgreater than or equal to about 10⁴ M⁻¹, more preferably of greater thanor equal to about 10⁵ M⁻¹, more preferably of greater than or equal toabout 10⁶ M⁻¹, and still more preferably of greater than or equal toabout 10⁷ M⁻¹. Affinities of binding partners or antibodies can bereadily determined using conventional techniques, for example, thosedescribed by Scatchard et al. (Ann. N.Y. Acad. Sci. USA 51:660 (1949))or by surface plasmon resonance (BIAcore, Biosensor, Piscataway, N.J.)(Wolff et al., Cancer Res. 53:2560-65 (1993).

Antibodies may generally be prepared by any of a variety of techniquesknown to those having ordinary skill in the art. See, e.g., Harlow etal., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory(1988). In one such technique, an animal is immunized with an NRG4polypeptide as an antigen to generate polyclonal antisera. Suitableanimals include, for example, rabbits, sheep, goats, pigs, cattle, andmay also include smaller mammalian species, such as mice, rats, andhamsters, or other species.

Human monoclonal antibodies may be generated by any number of techniqueswith which those having ordinary skill in the art will be familiar. Suchmethods include but are not limited to, Epstein Barr Virus (EBV)transformation of human peripheral blood cells (e.g., containing Blymphocytes) (see, e.g., U.S. Pat. No. 4,464,456); in vitro immunizationof human B cells (Boerner et al., J. Immunol. 147:86-95 (1991)); fusionof spleen cells from immunized transgenic mice carrying humanimmunoglobulin genes inserted by yeast artificial chromosomes (YAC)(U.S. Pat. No. 5,877,397; Bruggemann et al., Curr. Opin. Biotechnol.8:455-58 (1997); Jakobovits et al., Ann. N. Y. Acad. Sci. 764:525-35(1995)); isolation from human immunoglobulin V region phage libraries(U.S. Pat. No. 5,223,409; Huse et al., Science 246:1275-81 (1989), Kanget al., Proc. Natl. Acad. Sci. USA 88:4363-66 (1991); Hoogenboom et al.,J. Molec. Biol. 227:381-88 (1992); Schlebusch et al., Hybridoma 16:47-52(1997) and references cited therein), or other procedures as known inthe art and based on the disclosure herein.

Chimeric antibodies, specific for an NRG4 polypeptide, includinghumanized antibodies, may also be generated according to the presentinvention. A chimeric antibody has at least one constant region domainderived from a first mammalian species and at least one variable regiondomain derived from a second, distinct mammalian species. See, e.g.,Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-55 (1984). Inpreferred embodiments, a chimeric antibody may be constructed by cloningthe polynucleotide sequence that encodes at least one variable regiondomain derived from a non-human monoclonal antibody, such as thevariable region derived from a murine, rat, or hamster monoclonalantibody, into a vector containing a nucleic acid sequence that encodesat least one human constant region. See, e.g., Shin et al., MethodsEnzymol. 178:459-76 (1989); Walls et al., Nucleic Acids Res. 21:2921-29(1993). Another method known in the art for generating chimericantibodies is homologous recombination (e.g., U.S. Pat. No. 5,482,856).Preferably, the vectors will be transfected into eukaryotic cells forstable expression of the chimeric antibody.

A non-human/human chimeric antibody may be further geneticallyengineered to create a “humanized” antibody. Such a humanized antibodymay comprise a plurality of CDRs derived from an immunoglobulin of anon-human mammalian species, at least one human variable frameworkregion, and at least one human immunoglobulin constant region.Humanization may in certain embodiments provide an antibody that hasdecreased binding affinity for an NRG4 polypeptide when compared, forexample, with either a non-human monoclonal antibody from which an NRG4polypeptide binding variable region is obtained, or a chimeric antibodyhaving such a V region and at least one human C region, as describedabove. See, e.g., Jones et al., Nature 321:522-25 1(986); Riechmann etal., Nature 332:323-27 (1988); Padlan et al., FASEB 9:133-39 (1995);Chothia et al., Nature, 342:377-383 (1989); Bajorath et al., Ther.Immunol. 2:95-103 (1995); EP-0578515-A3.

Within certain embodiments, the use of antigen-binding fragments ofantibodies may be preferred. Such fragments include Fab fragments orF(ab′)₂ fragments, which may be prepared by proteolytic digestion withpapain or pepsin, respectively. The antigen binding fragments may beseparated from the Fc fragments by affinity chromatography, for example,using immobilized protein A or protein G, or immobilized NRG4polypeptide, or a suitable variant or fragment thereof. Those havingordinary skill in the art can routinely and without undueexperimentation determine what is a suitable variant or fragment basedon characterization of affinity purified antibodies obtained, forexample, using immunodetection methods as provided herein. Analternative method to generate Fab fragments includes mild reduction ofF(ab′)₂ fragments followed by alkylation. See, e.g., Weir, Handbook ofExperimental Immunology, Blackwell Scientific, Boston (1986).

According to certain embodiments, non-human, human, or humanized heavychain and light chain variable regions of any of the above-described Igmolecules may be constructed as single chain Fv (sFv) polypeptidefragments (single chain antibodies). See, e.g., Bird et al., Science242:423-26 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-83(1988).

An NRG4-binding immunoglobulin (or fragment thereof) as described hereinmay contain a detectable moiety or label such as an enzyme, cytotoxicagent, or other reporter molecule, including a dye, radionuclide,luminescent group, fluorescent group, or biotin, or the like. TheNRG4-specific immunoglobulin or fragment thereof may be radiolabeled fordiagnostic or therapeutic applications. Techniques for radiolabeling ofantibodies are known in the art. See, e.g., Adams, In Vivo 12:11-21(1998); Hiltunen, Acta Oncol. 32:831-39 (1993). Therapeutic applicationsare described in greater detail below and may include use of theNRG4-binding antibody (or fragment thereof) in conjunction with othertherapeutic agents. The antibody or fragment may also be conjugated to acytotoxic agent as known in the art and provided herein, for example, atoxin, such as a ribosome-inactivating protein, a chemotherapeuticagent, an anti-mitotic agent, an antibiotic or the like.

The invention also contemplates the generation of anti-idiotypeantibodies that recognize an antibody (or antigen-binding fragmentthereof) that specifically binds to an NRG4 polypeptide as providedherein, or a variant or fragment thereof. Anti-idiotype antibodies maybe generated as polyclonal antibodies or as monoclonal antibodies by themethods described herein, using an anti-NRG4 antibody (orantigen-binding fragment thereof) as immunogen. Anti-idiotype antibodiesor fragments thereof may also be generated by any of the recombinantgenetic engineering methods described above, or by phage displayselection. An anti-idiotype antibody may react with the antigen bindingsite of the anti-NRG4 antibody such that binding of the anti-NRG4antibody to an NRG4 polypeptide is competitively inhibited.Alternatively, an anti-idiotype antibody as provided herein may notcompetitively inhibit binding of an anti-NRG4 antibody to an NRG4polypeptide.

As provided herein and according to methodologies well known in the art,polyclonal and monoclonal antibodies may be used for the affinityisolation of NRG4 polypeptides. See, e.g., Hermanson et al., ImmobilizedAffinity Ligand Techniques, Academic Press, Inc. New York (1 992).Briefly, an antibody (or antigen-binding fragment thereof) may beimmobilized on a solid support material, which is then contacted with asample comprising the polypeptide of interest (e.g., an NRG4polypeptide). Following separation from the remainder of the sample, thepolypeptide is then released from the immobilized antibody.

Antibodies to NRG4 may find use in treatment of patients with coloncancer, in view of the upregulation of NRG4 in colon cancer cell linesas disclosed herein. Antibodies to NRG4 may also find use in modulatingeffects of NRG4 that are mediated through binding of NRG4 to itsreceptor, erbB-4.

Therapeutic and Diagnostic Uses of NRG4.

Members of the neuregulin family influence cellular proliferation,differentiation, and apoptosis. For example, neuregulin-1 stimulatesmitogenesis of mouse fibroblasts, human Schwann cells, epithelial andglial cells (Carraway et al., J. Biol. Chem. 270:7111-7116, 1995;Morrissey et al., Proc. Natl. Acad. Sci. U.S.A. 92:1431-1435,1995; Pelesand Yarden, Bioessays 15:815-824, 1993). Neuregulin-1 also acts as asurvival factor for astrocytes (Pinkas-Kramarski et al., Proc. Natl.Acad. Sci. U.S.A. 91:9387-9391, 1994). Thus, neuregulins regulate cellgrowth and tissue homeostasis and represent important targets fortreatment of diseases associated with aberrant cell growth, such ascancer, tumor progression, hyperproliferative cell growth oraccompanying biological or physical manifestations. NRG4 is a neuregulinfamily member which regulates cell proliferation.

Neuregulins are expressed in tissue- and stage-specific patterns.Neuregulin-1 isoforms are primarily expressed in neural and muscletissue (discussed in Carraway, Bioessays 18:263-266, 1996). According tothe present invention, real time polymerase chain reaction (PCR)analysis revealed that NRG4 is expressed at an elevated level inapproximately 50% of the colon/colorectal cell lines tested compared tonormal colon. In addition, of the normal tissues tested, all exceptpancreas and skeletal muscle demonstrated low levels of NRG4. Thus,according to the invention, NRG4 is likely to play a role in coloncancer.

Colon cancer may be treated using inhibitors or other modulators ofNRG4. Alternatively or in addition, NRG4 may be used as adifferentiation factor to treat the cancer directly.

The present invention includes methods of treating patients in needthereof with NRG4-encoding polynucleotides and NRG4 polypeptides. NRG4was downregulated in five pancreatic cancer cell lines. Thus, diseasesand disorders which can be treated using compositions of the presentinvention include, but are not limited to, cancers including pancreaticcancer.

The present invention includes therapeutic or pharmaceuticalcompositions comprising NRG4-encoding polynucleotides and NRG4polypeptides. Pharmaceutical compositions can comprise polypeptides,antibodies, small molecules or polynucleotides. Therapeutics, whetherpolynucleotide or polypeptide or small molecule, can be tested, forexample, in animal models and cell lines disclosed in Bosland,Encyclopedia of Cancer, volume II, pages 1283 to 1296 and 1303 to 1313(1997) by Academic Press. Pharmaceutical compositions may be designed toeither decrease or increase NRG4 activity, depending on the type ofdisease or disorder being treated. For example, cancers or diseasesassociated with hyperproliferation may be treated by reducing NRG4activity, for example, by administering therapeutically effectiveamounts of antisense NRG4 RNA, NRG4 ribozymes, RNAi specific for NRG4,inactivating antibodies, peptide or small molecule antagonists orinhibitors, NRG4 mutants or NRG4 receptors or mutants thereof. However,conditions or diseases that can be treated by promoting cellproliferation may be treated, for example, by administering biologicallyactive NRG4 polypeptides or an expression vector comprising NRG4polynucleotides encoding biologically active NRG4 polypeptides.

The term “therapeutically effective amount” as used herein refers to anamount of a therapeutic agent to treat, ameliorate, or prevent aspecific disease or condition, or to exhibit a detectable therapeutic orpreventive effect. The effect can be detected by, for example, chemicalmarkers or antigen levels. The effects also include reduction inphysical symptoms. The effective amount for a given situation can bedetermined by routine experimentation and is within the judgment of theclinician. The precise effective amount will vary depending on factorsincluding, but not limited to, the subject's size and health, the natureand extent of the condition, and the therapeutics selected foradministration. For purposes of the present invention, an effective dosewill be from about 0.05 mg/ kg to 50 mg/kg or 0.05 mg/kg to about 10mg/kg of the polynucleotide, polypeptide or antibody compositions in theindividual to which it is administered.

A pharmaceutical composition can also contain a pharmaceuticallyacceptable carrier. The term “pharmaceutically acceptable carrier”refers to a carrier for administration of a therapeutic agent, such asantibodies, polypeptides, polynucleotides and other therapeutic agents.Suitable carriers and pharmaceutically acceptable salts are well knownto those of ordinary skill in the art. A thorough discussion ofpharmaceutically acceptable excipients is available in Remington'sPharmaceutical Sciences (Mack Pub. Co., N.J. 1991).

Once formulated, the polynucleotide and polypeptide compositions of theinvention can be (1) administered directly to the subject; (2) deliveredex vivo, to cells derived from the subject; or (3) delivered in vitrofor expression of recombinant proteins. Direct delivery of thecompositions will generally be accomplished by injection,subcutaneously, intraperitoneally, intravenously, intra-arterially orintramuscularly, or delivered to the interstitial space of a tissue. Thecompositions can also be administered into a tumor or lesion. Othermodes of administration include oral and pulmonary administration,suppositories, and transdermal applications, needles, and gene guns orhyposprays. Dosage treatment may be a single dose schedule or a multipledose schedule.

Methods for the ex vivo delivery and reimplantation of transformed cellsinto a subject are known in the art and described in, e.g.,International Publication No. WO 93/14778. Generally, delivery ofnucleic acids for both ex vivo and in vitro applications can beaccomplished by, for example, dextran-mediated transfection, calciumphosphate precipitation transfection, viral infection, polybrenemediated transfection, protoplast fusion, electroporation, encapsulationof the polynucleotide(s) in liposomes, and direct microinjection of theDNA into nuclei, all well known in the art.

Examples of polynucleotide therapeutic agents include ribozymes,antisense RNA, and mammalian expression vectors. Trans-cleavingcatalytic RNAs (ribozymes) are RNA molecules possessing endoribonucleaseactivity. Ribozymes are engineered to cleave any RNA speciessite-specifically in the background of cellular RNA. The cleavage eventrenders the target RNA unstable and prevents protein expression.Ribozyme design and therapeutic uses are disclosed in Usman et al.,Current Opin. Struct. Biol. 6:527-533, 1996, which is incorporated byreference. The NRG4 polynucleotide sequence provides adequate sequencefor constructing an effective ribozyme. A target cleavage site isselected in the target sequence, and a ribozyme is constructed based onthe 5′ and 3′ nucleotide sequences that flank the cleavage site.Retroviral vectors are engineered to express monomeric and multimerichammerhead ribozymes targeting the mRNA of the NRG4 polynucleotidecoding sequence. These monomeric and multimeric ribozymes are tested invitro for an ability to cleave the NRG4 mRNA. A cell line is stablytransduced with the retroviral vectors expressing the ribozymes, and thetransduction is confirmed by Northern blot analysis andreverse-transcription polymerase chain reaction (RT-PCR). The cells arescreened for inactivation of the target mRNA by such indicators asreduction of expression of disease markers or reduction of the geneproduct of the target mRNA.

The present invention also relates to antisense oligonucleotidesdesigned to interfere with the normal function of NRG4 polynucleotides.Any modifications or variations of the antisense molecule which areknown in the art to be broadly applicable to antisense technology areincluded within the scope of the invention. Such modifications includepreparation of phosphothioate linkages, with increased stability, asdisclosed in U.S. Pat. Nos. 5,536,821; 5,541,306; 5,550,111; 5,563,253;5,571,799; 5,587,361, 5,625,050 and 5,958,773.

The antisense compounds of the invention can include modified bases asdisclosed in U.S. Pat. No. 5,958,773 and patents disclosed therein. Theantisense oligonucleotides of the invention can also be modified bychemically linking the oligonucleotide to one or more moieties orconjugates to enhance the activity, cellular distribution, or cellularuptake of the antisense oligonucleotide. Such moieties or conjugatesinclude lipids such as cholesterol, cholic acid, thioether, aliphaticchains, phospholipids, polyamines, polyethylene glycol (PEG), palmitylmoieties, and others as disclosed in, for example, U.S. Pat. Nos.5,514,758, 5,565,552, 5,567,810, 5,574,142, 5,585,481, 5,587,371,5,597,696 and 5,958,773.

Chimeric antisense oligonucleotides are also within the scope of theinvention, and can be prepared from the present inventiveoligonucleotides using the methods described in, for example, U.S. Pat.Nos. 5,013,830, 5,149,797, 5,403,711, 5,491,133, 5,565,350, 5,652,355,5,700,922 and 5,958,773.

In the antisense art a certain degree of routine experimentation isrequired to select optimal antisense molecules for particular targets.To be effective, the antisense molecule preferably is targeted to anaccessible, or exposed, portion of the target RNA molecule. Although insome cases information is available about the structure of target mRNAmolecules, the current approach to inhibition using antisense is viaexperimentation. The level of the target mRNA in the cell can bemeasured routinely in treated and control cells by reverse transcriptionof the mRNA with or without PCR amplification and assaying the resultingcDNA levels. The biological effect of antisense delivery to cells can bedetermined routinely by measuring cell growth or viability as is knownin the art.

Measuring the specificity of antisense activity by generating, assayingand analyzing cDNA levels is an art-recognized method of validatingantisense results. For example, RNA from treated and control cells canbe reverse-transcribed and the resulting cDNA populations analyzed.(Branch, A. D., T.I.B.S. 23:45-50, 1998.).

Antisense nucleic acids are designed to specifically bind to RNA,resulting in the formation of RNA-DNA or RNA-RNA hybrids, with an arrestof DNA replication, reverse transcription or messenger RNA translation.Antisense polynucleotides based on a selected sequence can interferewith expression of the corresponding gene. Antisense polynucleotides aretypically generated within the cell by expression from antisenseconstructs that contain the antisense strand as the transcribed(product) strand. Antisense polynucleotides can bind and/or interferewith the translation of the corresponding mRNA. The expression productsof control cells and cells treated with the antisense construct arecompared to detect the effects of the antisense polynucleotide on theprotein product of the gene corresponding to the polynucleotide. Theprotein is detected and identified using routine biochemical methods.

Antisense therapy for a variety of cancers is in clinical phase and hasbeen discussed extensively in the literature. Given the extensivebackground literature and clinical experience in antisense therapy, oneskilled in the art can use antisense NRG4 polynucleotides astherapeutics. The dosage and means of administration are determinedbased on the specific qualities of the composition, the patient, theprogression of the disease and other relevant factors. Preferably, thetherapeutic antisense composition contains an expression constructcomprising a promoter upstream of a polynucleotide segment of at least12, 18, 20, 22, 25, 30, or 35 contiguous nucleotides of NRG4 in theantisense orientation. Therapeutic antisense agents may be administeredlocally or systemically by a variety of methods known in the art.Examples cited include those mentioned above and receptor-mediatedtargeted delivery.

Therapeutic compositions containing antisense NRG4 polynucleotides areadministered in a range of about 100 ng to about 200 mg ofpolynucleotides for local administration in a gene therapy protocol, asdiscussed later. In all cases, routine experimentation in clinicaltrials will determine specific ranges for optimal therapeutic effects.

The invention also contemplates introduction of RNA with partial orfully double-stranded character into the cell or into the extracellularenvironment. Inhibition is specific to the NRG4 expression in that anucleotide sequence from a portion of the target NRG4 gene is chosen toproduce inhibitory RNA. This process is (1) effective in producinginhibition of gene expression, and (2) specific to the targeted NRG4gene. The procedure may provide partial or complete loss of function forthe target NRG4 gene. A reduction or loss of gene expression in at least99% of other targeted cells has been shown using such methods. Incertain embodiments, the target cell containing the target NRG4 gene maybe a pancreatic cell of a human, or a pancreatic tumor cell of a human.Lower doses of injected material and longer times after administrationof dsRNA may result in inhibition in a smaller fraction of cells.Quantitation of gene expression in a cell may show similar amounts ofinhibition at the level of accumulation of target mRNA or translation oftarget protein. Methods of preparing and using RNAi are generallydisclosed in U.S. Pat. No. 6,506,559, incorporated herein by reference.

The RNA may comprise one or more strands of polymerized ribonucleotide;it may include modifications to either the phosphate-sugar backbone orthe nucleoside. The double-stranded structure may be formed by a singleself-complementary RNA strand or two complementary RNA strands. RNAduplex formation may be initiated either inside or outside the cell. TheRNA may be introduced in an amount which allows delivery of at least onecopy per cell. Higher doses of double-stranded material may yield moreeffective inhibition. Inhibition is sequence-specific in that nucleotidesequences corresponding to the duplex region of the RNA are targeted forgenetic inhibition. RNA containing a nucleotide sequence identical to aportion of the NRG4 target gene is preferred for inhibition. RNAsequences with insertions, deletions, and single point mutationsrelative to the target sequence have also been found to be effective forinhibition. Thus, sequence identity may be optimized by alignmentalgorithms known in the art and calculating the percent differencebetween the nucleotide sequences. Alternatively, the duplex region ofthe RNA may be defined functionally as a nucleotide sequence that iscapable of hybridizing with a portion of the target gene transcript.

RNA may be synthesized either in vivo or in vitro. Endogenous RNApolymerase of the cell may mediate transcription in vivo, or cloned RNApolymerase can be used for transcription in vivo or in vitro. Fortranscription from a transgene in vivo or an expression construct, aregulatory region may be used to transcribe the RNA strand (or strands).

For RNAi, the RNA may be directly introduced into the cell (i.e.,intracellularly); or introduced extracellularly into a cavity,interstitial space, into the circulation of an organism, introducedorally, or may be introduced by bathing an organism in a solutioncontaining RNA. Methods for oral introduction include direct mixing ofRNA with food of the organism, as well as engineered approaches in whicha species that is used as food is engineered to express an RNA, then fedto the organism to be affected. Physical methods of introducing nucleicacids include injection directly into the cell or extracellularinjection into the organism of an RNA solution.

The advantages of the method include the ease of introducingdouble-stranded RNA into cells, the low concentration of RNA which canbe used, the stability of double-stranded RNA, and the effectiveness ofthe inhibition.

Inhibition of gene expression refers to the absence (or observabledecrease) in the level of protein and/or mRNA product from a NRG4 targetgene. Specificity refers to the ability to inhibit the target genewithout manifest effects on other genes of the cell. The consequences ofinhibition can be confirmed by examination of the outward properties ofthe cell or organism or by biochemical techniques such as RNA solutionhybridization, nuclease protection, Northern hybridization, reversetranscription, gene expression monitoring with a microarray, antibodybinding, enzyme linked immunosorbent assay (ELISA), Western blotting,radioimmunoassay (RIA), other immunoassays, and fluorescence activatedcell analysis (FACS). For RNA-mediated inhibition in a cell line orwhole organism, gene expression is conveniently assayed by use of areporter or drug resistance gene whose protein product is easilyassayed. Such reporter genes include acetohydroxyacid synthase (AHAS),alkaline phosphatase (AP), beta galactosidase (LacZ), beta glucoronidase(GUS), chloramphenicol acetyltransferase (CAT), green fluorescentprotein (GFP), horseradish peroxidase (HRP), luciferase (Luc), nopalinesynthase (NOS), octopine synthase (OCS), and derivatives thereof.Multiple selectable markers are available that confer resistance toampicillin, bleomycin, chloramphenicol, gentamycin, hygromycin,kanamycin, lincomycin, methotrexate, phosphinothricin, puromycin, andtetracyclin.

Depending on the assay, quantitation of the amount of gene expressionallows one to determine a degree of inhibition which is greater than10%, 20%, 30%, 50%, 60%, 70%, 80%, 90%, 95% or 99% as compared to a cellnot treated according to the present invention. Lower doses of injectedmaterial and longer times after administration of dsRNA may result ininhibition in a smaller fraction of cells (e.g., at least 10%, 20%, 50%,75%, 90%, or 95% of targeted cells). Quantitation of NRG4 geneexpression in a cell may show similar amounts of inhibition at the levelof accumulation of NRG4 target mRNA or translation of NRG4 targetprotein. As an example, the efficiency of inhibition may be determinedby assessing the amount of gene product in the cell: mRNA may bedetected with a hybridization probe having a nucleotide sequence outsidethe region used for the inhibitory double-stranded RNA, or translatedpolypeptide may be detected with an antibody raised against thepolypeptide sequence of that region.

The RNA may comprise one or more strands of polymerized ribonucleotide.It may include modifications to either the phosphate-sugar backbone orthe nucleoside. For example, the phosphodiester linkages of natural RNAmay be modified to include at least one of a nitrogen or sulfurheteroatom. Modifications in RNA structure may be tailored to allowspecific genetic inhibition while avoiding a general panic response insome organisms which is generated by dsRNA. Likewise, bases may bemodified to block the activity of adenosine deaminase. RNA may beproduced enzymatically or by partial/total organic synthesis, anymodified ribonucleotide can be introduced by in vitro enzymatic ororganic synthesis.

The double-stranded structure may be formed by a singleself-complementary RNA strand or two complementary RNA strands. RNAduplex formation may be initiated either inside or outside the cell. TheRNA may be introduced in an amount which allows delivery of at least onecopy per cell. Higher doses (e.g., at least 5, 10, 100, 500 or 1000copies per cell) of double-stranded material may yield more effectiveinhibition; lower doses may also be useful for specific applications.Inhibition is sequence-specific in that nucleotide sequencescorresponding to the duplex region of the RNA are targeted for geneticinhibition.

RNA containing a nucleotide sequences identical to a portion of the NRG4target gene are preferred for inhibition. RNA sequences with insertions,deletions, and single point mutations relative to the target sequencemay be effective for inhibition. Thus, sequence identity may optimizedby sequence comparison and alignment algorithms known in the art (seeGribskov and Devereux, Sequence Analysis Primer, Stockton Press, 1991,and references cited therein) and calculating the percent differencebetween the nucleotide sequences by, for example, the Smith-Watermanalgorithm as implemented in the BESTFIT software program using defaultparameters (e.g., University of Wisconsin Genetic Computing Group).Greater than 90% sequence identity, or even 100% sequence identity,between the inhibitory RNA and the portion of the NRG4 target gene ispreferred. Alternatively, the duplex region of the RNA may be definedfunctionally as a nucleotide sequence that is capable of hybridizingwith a portion of the NRG4 target gene transcript (e.g., 400 mM NaCl, 40mM PIPES pH 6.4, 1 mM EDTA, 50° C. or 70° C. hybridization for 12-16hours; followed by washing). The length of the identical nucleotidesequences may be at least 25, 50, 100, 200, 300 or 400 bases.

100% sequence identity between the RNA and the NRG4 target gene is notrequired to practice the present invention. Thus the methods have theadvantage of being able to tolerate sequence variations that might beexpected due to genetic mutation, strain polymorphism, or evolutionarydivergence.

NRG4 RNA may be synthesized either in vivo or in vitro. Endogenous RNApolymerase of the cell may mediate transcription in vivo, or cloned RNApolymerase can be used for transcription in vivo or in vitro. Fortranscription from a transgene in vivo or an expression construct, aregulatory region (e.g., promoter, enhancer, silencer, splice donor andacceptor, polyadenylation) may be used to transcribe the RNA strand (orstrands). Inhibition may be targeted by specific transcription in anorgan, tissue, or cell type; stimulation of an environmental condition(e.g., infection, stress, temperature, chemical inducers); and/orengineering transcription at a developmental stage or age. The RNAstrands may or may not be polyadenylated; the RNA strands may or may notbe capable of being translated into a polypeptide by a cell'stranslational apparatus.

RNA may be chemically or enzymatically synthesized by manual orautomated reactions. The RNA may be synthesized by a cellular RNApolymerase or a bacteriophage RNA polymerase (e.g., T3, T7, SP6). Theuse and production of an expression construct are known in the art (forexample, WO 97/32016; U.S. Pat. Nos. 5,593,874, 5,698,425, 5,712,135,5,789,214, and 5,804,693; and the references cited therein). Ifsynthesized chemically or by in vitro enzymatic synthesis, the RNA maybe purified prior to introduction into the cell. For example, RNA can bepurified from a mixture by extraction with a solvent or resin,precipitation, electrophoresis, chromatography, or a combinationthereof. Alternatively, the RNA may be used with no or a minimum ofpurification to avoid losses due to sample processing. The RNA may bedried for storage or dissolved in an aqueous solution. The solution maycontain buffers or salts to promote annealing, and/or stabilization ofthe duplex strands.

RNA may be directly introduced into the cell (i.e., intracellularly); orintroduced extracellullady into a cavity, interstitial space, into thecirculation of an organism, introduced orally, or may be introduced bybathing an organism in a solution containing the RNA. Methods for oralintroduction include direct mixing of the RNA with food of the organism,as well as engineered approaches in which a species that is used as foodis engineered to express the RNA, then fed to the organism to beaffected. For example, the RNA may be sprayed onto a plant or a plantmay be genetically engineered to express the RNA in an amount sufficientto kill some or all of a pathogen known to infect the plant. Physicalmethods of introducing nucleic acids, for example, injection directlyinto the cell or extracellular injection into the organism, may also beused. Vascular or extravascular circulation, the blood or lymph system,and the cerebrospinal fluid are sites where the RNA may be introduced. Atransgenic organism that expresses RNA from a recombinant construct maybe produced by introducing the construct into a zygote, an embryonicstem cell, or another multipotent cell derived from the appropriateorganism.

Physical methods of introducing nucleic acids include injection of asolution containing the RNA, bombardment by particles covered by theRNA, soaking the cell or organism in a solution of the RNA, orelectroporation of cell membranes in the presence of the RNA. A viralconstruct packaged into a viral particle would accomplish both efficientintroduction of an expression construct into the cell and transcriptionof RNA encoded by the expression construct. Other methods known in theart for introducing nucleic acids to cells may be used, such aslipid-mediated carrier transport, chemical-mediated transport, such ascalcium phosphate, and the like. Thus the RNA may be introduced alongwith components that perform one or more of the following activities:enhance RNA uptake by the cell, promote annealing of the duplex strands,stabilize the annealed strands, or other-wise increase inhibition of thetarget gene.

The RNAi may be used alone or as a component of a kit having at leastone of the reagents necessary to carry out the in vitro or in vivointroduction of RNA to test samples or subjects. Preferred componentsare the dsRNA and a vehicle that promotes introduction of the dsRNA.Such a kit may also include instructions to allow a user of the kit topractice the invention.

Suitable injection mixes are constructed so animals receive an averageof 0.5×10⁶ to 1.0×10⁶ molecules of RNA. For comparisons of sense,antisense, and dsRNA activities, injections are compared with equalmasses of RNA (i.e., dsRNA at half the molar concentration of the singlestrands). Numbers of molecules injected per adult are given as roughapproximations based on concentration of RNA in the injected material(estimated from ethidium bromide staining) and injection volume(estimated from visible displacement at the site of injection). Avariability of several-fold in injection volume between individualanimals is possible.

Polypeptide compositions can also include antibodies and peptides. Theeffective dosages, for therapeutic compositions containing protein,polypeptide or antibody are in the range of about 10 μg to about 50μg/kg of patient body weight, about 50 μg to about 5 mg/kg, about 100 μgto about 500 μg/kg of patient body weight, and about 200 to about 250μg/kg.

Antibodies may be polyclonal, monoclonal or single-chain antibodiesprepared by methods known in the field. Antibodies specific to NRG4polypeptides bind the protein and inhibit the protein from functioningin the cell. For example, the antibodies can prevent NRG4 from bindingto binding partners or receptors. The invention also pertains toantibodies directed against protein partners and receptors for NRG4polypeptides. Such antibodies can disrupt protein:protein interactionsrequired for cellular function of NRG4. They can also inhibit downstreamsignaling by the receptor protein.

Therapeutic compositions and methods comprising peptide-agonists andantagonists are also included in the invention. The peptides can affectthe function of polypeptides encoded by NRG4 mRNA or their bindingpartners and receptors. For example, the peptides may blockprotein:protein interactions or cause conformational changes whichdiminish or enhance NRG4's normal functional activity. The peptides mayalso alter NRG4's activity or specificity in a therapeutically usefulmanner.

The therapeutic polynucleotides of the present invention may be utilizedin gene delivery vehicles. The gene delivery vehicle may be of viral ornon-viral origin (see generally, Jolly, Cancer Gene Therapy 1:51-64,1994; Kimura, Human Gene Therapy 5:845-852, 1994; Connelly, Human GeneTherapy 1:185-193, 1995; and Kaplitt, Nature Genetics 6:148-153, 1994).Gene therapy vehicles for delivery of constructs including a codingsequence of NRG4 can be administered either locally or systemically.Expression of such coding sequences can be driven using endogenousmammalian or heterologous promoters. Expression of the coding sequencecan be either constitutive or regulated.

Any gene delivery method known in the art can be utilized. For example,the present invention can employ recombinant retroviruses that areconstructed to carry or express a selected nucleic acid molecule ofinterest. The present invention also employs alphavirus-based vectorsand parvovirus, which can function as gene delivery vehicles. Other genedelivery vehicles and methods may be employed, including polycationiccondensed DNA linked or unlinked to killed adenovirus alone, for exampleCuriel, Hum. Gene Ther. 3:147-154, 1992; ligand linked DNA, for examplesee Wu, J. Biol. Chem. 264:16985-16987, 1989; eukaryotic cell deliveryvehicles, for example see U.S. Ser. No. 08/240,030, filed May 9, 1994,and U.S. Pat. No. 6,015,686; deposition of photopolymerized hydrogelmaterials; hand-held gene transfer particle gun, as described in U.S.Pat. No. 5,149,655; ionizing radiation as described in U.S. Pat. No.5,206,152 and in WO92/11033; nucleic charge neutralization or fusionwith cell membranes. Additional approaches are described in Philip, Mol.Cell Biol. 14:2411-2418, 1994, and in Woffendin, Proc. Natl. Acad. Sci.91:11581-11585, 1994. Packaging cell lines suitable for use with theabove-described retroviral vector constructs may be readily prepared(see PCT publications WO 95/30763 and WO 92/05266), and used to createproducer cell lines (also termed vector cell lines) for the productionof recombinant vector particles.

Non-viral delivery methods include, but are not limited to, mechanicaldelivery systems such as the approach described in Woffendin et al.,Proc. Natl. Acad. Sci. USA 91(24):11581-11585,1994, and naked DNAprotocols. Exemplary naked DNA introduction methods are described in WO90/11092 and U.S. Pat. No. 5,580,859.

Subgenomic NRG4 polynucleotides and complements thereof and NRG4antibodies can be used as markers to diagnose and determine theprognosis of cancer, tumor progression, hyperproliferative cell growthor accompanying biological and physical manifestations. Levels of NRG4polynucleotides or polypeptides in a sample are compared to the levelsin a normal control sample. The normal sample can include a pool ofcells from a particular tissue or tissues and/or cells from throughoutthe body. Immunoassays or nucleic acid assays can be used for suchmeasurements. Any observed difference between the sample and normalcontrol can indicate the occurrence of disease or disorder. Typically,if the levels of NRG4 polynucleotides or polypeptides are higher orlower than those found in the normal control, the results can indicatethe occurrence of cancer, tumor progression, hyperproliferative cellgrowth and/or accompanying biological or physical manifestations.

Nucleic acid assays utilize subgenomic polynucleotides capable ofhybridizing under stringent conditions to NRG4 polynucleotides orcomplements thereof. Polynucleotide probes comprising at least 10, 25,50 contiguous nucleotides or more selected from the nucleotide sequenceof NRG4 are labeled, for example, with a radioactive, fluorescent,biotinylated, or chemiluminescent label, and detected by well knownmethods appropriate for the particular label selected. Subgenomicpolynucleotides are preferably intron-free. Polynucleotidescorresponding to NRG4 can be introduced into vectors and propagated insuitable hosts. Plasmids can be introduced into host cells usingtechniques available in the art. These techniques include, but are notlimited to, electroporation and calcium phosphate-mediated transfection.They can be isolated and purified from DNA vectors by standardtechniques, such as restriction enzyme digestion and gel electrophoresisor chromatography. The polynucleotides can also be produced using thepolymerase chain reaction according to techniques well known in the art.

By “stringent conditions” is meant, for example, hybridizationstringency conditions of wash conditions with 2×SSC, 0.1% SDS, roomtemperature twice, 30 minutes each; then 2×SSC, 0.1% SDS, 50° C. once,30 minutes; then 2×SCC, room temperature twice, 10 minutes each.Hybridization is affected by a variety of parameters includingtemperature, salt concentration, formamide concentration, and length ofprobe. Some guidelines regarding hybridization conditions are asfollows: (a) the higher the temperature, the more specific thehybridization; (b) lower salt concentrations mean higher stringency atany one temperature; (c) formamide reduces annealing temperature byabout 0.62° C. for every 1% increase in formamide concentration; and (d)the longer the probe, the less specific hybridization. Table 2 of U.S.Pat. No. 6,440,683 discloses examples of stringency conditions. One ofskill in the art will be familiar with using comparable stringencyconditions to evaluate and compare the formation of complexes between asample nucleotide of a tissue suspected of being diseased (for example,colon cancer or pancreatic cancer) or a normal sample, and a nucleotidesuch as, for example, SEQ ID NO:1, and a fragment thereof, such as aprobe.

A further reference for hybridization conditions is Sambrook, J. et al,1989, Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., Chapter 9, as known to thoseof skill in the art.

The subgenomic polynucleotides can be used to compare related genes innormal control tissue and suspected diseased tissue by any means knownin the art. For example, the NRG4 gene or mRNA from a suspected diseasedtissue can be sequenced and compared with the NRG4 gene or mRNA sequencein the normal tissue. The NRG4-related genes, or portions thereof, inthe two tissues are amplified, for example using nucleotide primersbased on the nucleotide sequence of NRG4, using the polymerase chainreaction. The amplified genes or mRNAs or portions thereof arehybridized to nucleotide probes selected from the same NRG4 nucleotidesequence and sequenced. A difference in the nucleotide sequence of theNRG4-related gene in the tissue suspected of being diseased comparedwith the normal nucleotide sequence suggests a role of the NRG4 proteinin the disease, and provides a lead for preparing a therapeutic agent.The nucleotide probes or nucleotides incorporated during sequencing arelabeled by a variety of methods, such as radiolabeling, biotinylation,or labeling with fluorescent or chemiluminescent tags, and detected bystandard methods known in the art.

Alternatively, NRG4 mRNA levels in normal and suspected diseased tissuesare compared. PolyA⁺ RNA is isolated from the two tissues as is known inthe art. For example, one of skill in the art can readily determinedifferences in the size or amount of NRG4-related mRNA transcriptsbetween the two tissues by Northern blot analysis, primer extension, S1nuclease protection, reverse transcription-polymerase chain reaction(RT-PCR), or in situ hybridization using polynucleotide probescorresponding to NRG4 or complement thereof. Increased or decreasedexpression of an NRG4-related mRNA in a tissue sample suspected of beingdiseased, compared with the expression of the same NRG4-related mRNA ina normal tissue, suggests that the NRG4 protein or lack thereof has arole in the disease, and also provides a lead for preparing atherapeutic agent.

NRG4 gene expression can also be examined using polynucleotide arrays.Polynucleotide arrays provide a high throughput technique that can assaya large number of polynucleotide sequences in a sample. This technologycan be used as a diagnostic and as a tool to test for differentialexpression of an encoded protein. Techniques for constructing arrays andmethods of using these arrays are described in EP No. 0 799 897; PCT.No.WO 97/29212; PCT No. WO 97/27317; EP No. 0 785 280; PCT No. WO 97/02357;U.S. Pat. No. 5,593,839; U.S. Pat. No. 5,578,832; EP No. 0 728 520; U.S.Pat. No. 5,599,695; EP No. 0 721 016; U.S. Pat. No. 5,556,752; PCT No.WO 95/22058; and U.S. Pat. No. 5,631,734, which are incorporated byreference.

Antibodies which bind NRG4 and/or variant polypeptides can be used indiagnosing and determining the prognosis of cancer, tumor progression,hyperproliferative cell growth or accompanying biological and physicalmanifestations. These antibodies may be monoclonal, polyclonal or singlechain antibodies and are produced by methods well known in the art.

Any method known in the art can be used to compare NRG4 proteins fromnormal control samples and suspected diseased samples. The size of theprotein in the two tissues can be compared, for example, using anti-NRG4antibodies to detect NRG4 polypeptides by Western blot. Alterations inthe size of the NRG4 protein in a tissue suspected of being diseasedcompared with the size in a normal control sample indicate the proteinis abnormal, possibly due to truncation, deletion or alteredpost-translational modification. Size alterations are indicative thatNRG4 has a role in the disease and provide a lead for preparing atherapeutic agent. Other changes, such as protein expression levels andsubcellular localization can also be detected immunologically, forexample by using antibodies directed against NRG4 for Western blot orimmunofluorescence. A higher or lower level of NRG4 protein in a tissuesuspected of being diseased, or in conditioned media of cells derivedfrom said tissue, or in blood from a patient suspected of beingdiseased, compared with the level in a normal control sample, isindicative that NRG4 has a role in the disease and provides another leadfor preparing a therapeutic agent. Similarly, changes in subcellularlocalization of NRG4 protein also indicate NRG4 has a role in thedisease.

Reagents specific for NRG4 polynucleotides and polypeptides, such asantibodies and nucleotide probes, can be supplied in a kit for detectingthe presence of an expression product in a biological sample. The kitcan also contain buffers or labeling components, detection reagents, andinstructions for using reagents to detect and quantify expressionproducts in biological samples and control normal biological samples.Normal biological samples may be in any form suitable for the particularmethod of detection utilized by the kit. For example, normal biologicalsamples can be polynucleotides, polypeptides, cellular extracts ortissue sections.

Preferred embodiments of the invention are described in the followingexamples. Other embodiments within the scope of the claims herein willbe apparent to one skilled in the art from consideration of thespecification or practice of the invention as disclosed herein. It isintended that the specification, together with the examples, beconsidered exemplary only, with the scope and spirit of the inventionbeing indicated by the claims that follow the examples.

All patents and publications cited herein are incorporated by referenceherein.

EXAMPLES Example 1 Tissue Distribution of NRG4

In order to determine the tissue distribution of NRG4 (the DNA and aminoacid sequences for which are shown in SEQ ID NOs:1 and 2, respectively),quantitative PCR was performed on a number of normal and tumor cellslines, particularly colorectal and pancreatic cell lines. Briefly,quantitative real-time PCR was performed on either commerciallyavailable tissue RNA (Ambion, Stratagene) or on RNA isolated from thedesired cells using a Roche Isolation kit according to themanufacturer's instructions. One microgram of RNA was then used as atemplate to synthesize a first strand cDNA using MMLV reversetranscriptase (Ambion) and the manufacturer's buffer and recommendedconcentrations of oligo-deoxynucleotides (oligo dTs), nucleotides, andRNAsin. Specifically, for each 20 μl reaction, 1 μg of RNA was placedinto a sterile tube and water was added to a final volume of 12.5 μl. Toeach tube was added 7.5 μl of a buffer/enzyme mix, prepared by mixing2.5 μl H₂O, 2 μl 10× reaction PCR buffer (Ambion), 1 μl oligo dT (20pmol), 1 μl dNTP mix (10 mM each), 0.5 μl RNAsin (20 U) and 0.5 μl MMLVreverse transcriptase (50 U). The contents of the reaction were mixedand the reaction was incubated at 42° C. for 1 hour, then centrifugedprior to amplification.

The first stand cDNA generated as described above was then used as atemplate for quantitative real-time PCR using the GeneAmp 5700 SequenceDetection System (PE Biosystems) as recommended by the manufacturer.NRG4 sequences were amplified with the forward primer5′-ACAGATCACGMGAGCCCTGTGG-3′ (SEQ ID NO:3) and reverse primer5′-GGTACTGCTCGTCTCTACCAGGTTG-3′ (SEQ ID NO:4). Small differences inamounts of total template in the first-strand cDNA reaction wereeliminated by normalizing to the amount of actin or GusB sequencesamplified in separate quantitative PCR reactions. The forward primer foractin sequence amplification was 5′-CGGGAAATCGTGCGTGACATTMG-3′ (SEQ IDNO:5) and the reverse primer 5′-TGATCTCCTTCTGCATCCTGTCGG-3′ (SEQ IDNO:6). The forward primer for GusB sequence amplification was5′-CCTTTTGCGAGAGAGATACT-3′ (SEQ ID NO:7) and the reverse primer was5′-CCTTAGTGTTCCCTGCTAG-3′ (SEQ ID NO:8). An amplification mixture wasprepared by mixing in the following order: 8.7 μl water, 12.5 μl 2× SYBRgreen mix (Molecular Probes), 0.9 μl of each primer (5 pmol/μl each) and2 μl of template RT product reaction described in the precedingparagraph, bringing the total volume to 20 μl. Amplification on the PEBiosystems 5700 was carried out according to standard protocol. SYBRGreen (Molecular Probes, Eugene, Oreg.) is a dye that fluoresces whenbound to double stranded DNA. As double stranded PCR product isgenerated during amplification, the fluorescence from SYBR Greenincreases. PCR products were quantified based on the cycle at which theamplification entered the linear phase of amplification in comparison toan internal standard, and using software supplied by the manufacturer.

Real time PCR results demonstrated that approximately 50% of thecolon/colorectal cell lines examined exhibited up-regulation of NRG4mRNA when compared to normal colon. (FIG. 1, Table 2) Results alsorevealed down-regulation of NRG4 mRNA in 100% of pancreatic cancer celllines tested. (Table 1.) Of the normal tissues tested (testicle, thymus,skeletal muscle, small intestine, cervix, ovary, stomach, prostate,placenta, adrenal gland, liver, lung, brain, kidney, spleen, colon,pancreas, heart, bladder, and uterus) low level expression was only seenin testicle, skeletal muscle and pancreas. (FIG. 2.) TABLE 1 Expressionin pancreatic cancer cell lines NRG4 expression ATCC relative to GusBCell Line number Cell type expression Normal 14.5 pancreas AsPC-1CRL-1682 Adenocarcinoma 4.1 Capan1 HTB-79 Metastatic 3.2 adenocarcinomaMiaPaCa2 CRL-1420 Primary cell line 0.1 Panc1 CRL-1469 Primary cell line0.8 PL45 CRL-2558 Ductal adenocarcinoma 0.1

TABLE 2 Expression in colon cancer cell lines NRG4 expression ATCCrelative to GusB Cell line number Cell type expression Colon 0.9Colo320DM CCL-220 Adenocarcinoma 3.7 Km12C CMCC 11825 Colon-low 3.2metastatic potential Km12L4 CMCC 11611 Colon-high 4.45 metastaticpotential LoVo CCL-229 Colorectal carcinoma 0.1 HCT116 CCL-247Colorectal carcinoma 1.95

Example 2 Immunohistochemical Analysis of NRG4 Expression

To determine if there is differential expression of NRG4 protein incancer versus normal tissues, immunohistochemistry is performed on colonand pancreatic tissues from normal and cancer patients using monoclonaland/or polyclonal antibodies against NRG4.

In order to perform this analysis, the slides containingparaffin-embedded sections of interest are de-waxed and rehydrated withxylene followed by alcohol and phosphate-buffered saline (PBS), prior toantigen imaging. The slides are immersed in 1× Citra Plus Solution andplaced in a pressure cooker and heated for 5 minutes. After cooling, theslides are rinsed with deionized water and blocked in 3% H₂O₂ for 10minutes at room temperature. The slides are washed with deionized waterand PBS and blocked with avidin at room temperature for 10 minutes.Biotin is then applied to the slide for 10 minutes at room temperatureafter washing in PBS to remove the avidin. Post-biotin treatment, theslides are rinsed in PBS and immersed in protein block (1× PowerBlock)for 8 minutes at room temperature. After rinsing with PBS, 350 μl ofanti-NRG4 antibody or control antibody is applied on the section andincubated for 1 hour at room temperature. The slides are then washedthree times with PBS for 3-5 minutes per wash. The secondary link(BioGenex Multi-Link) is applied for 10 minutes at room temperature andthen washed 3 times with PBS.

To visualize the antigen, BioGenex HRP label is applied to the sectionsfor 10 minutes at room temperature and then removed by washing with PBS.BioGenex H₂O₂ substrate is added at room temperature for 10 minutes,followed by washing with deionized water. The sections are thencounterstained with hematoxylin for 1 minute at room temperature. Theslides are then rinsed twice with water, incubated in PBS for 1 minuteand rinsed once again with water to remove the PBS. A drop of BioGenexSuper Mount is applied to the section and air-dried overnight at roomtemperature. The slides are analyzed by microscopy for localization ofNRG4.

Example 3 Antisense Regulation of NRG4 Expression

Additional functional information on NRG4 is generated using antisenseknockout technology. NRG4 expression in cancerous cells is furtheranalyzed to confirm the role and function of NRG4 polypeptides intumorigenesis, e.g., in promoting a metastatic phenotype.

A number of different oligonucleotides complementary to NRG4 mRNA aredesigned as potential antisense oligonucleotides and tested for theirability to suppress expression of NRG4. The ability of each designedantisense oligonucleotide to inhibit gene expression is tested throughtransfection into SW620 or Km12L4 colon colorectal carcinoma cells. Foreach transfection mixture, a carrier molecule, preferably a lipitoid orcholesteroid, is prepared to a working concentration of 0.5 mM in water,sonicated to yield a uniform solution, and filtered through a 0.45 μmPVDF membrane. The antisense or control oligonucelotide is then preparedto a working concentration of 100 μM in sterile Millipore water. Theoligonucleotide is further diluted in OptiMEM™ (Gibco BRL), in amicrofuge tube, to 2 μM, or approximately 20 μg oligo/ml of OptiMEM™. Ina separate microfuge tube, lipitoid or cholesteroid, typically in theamount of about 1.5-2 nmol lipitoid/μg antisense oligonucleotide, isdiluted in the same volume of OptiMEM™ used to dilute theoligonucleotide. The diluted antisense oligonucleotide is thenimmediately added to the diluted lipitoid or cholesteroid and mixed bypipetting up and down. The oligonucleotide is then added to the cells toa final concentration of 30 nM.

The level of the NRG4 mRNA is quantitated in the transfected cancer celllines using the Roche LightCycler™ real-time PCR machine. Values for theNRG4 mRNA are normalized versus an internal control (e.g., beta-actin).For each 20 μl reaction, extracted RNA (generally 0.2-1 μg total) isplaced in a sterile 0.5 or 1.5 ml microcentrifuge tube, and water isadded to a total volume of 12.5 μl. To each tube is added 7.5 μl of abuffer/enzyme mixture, prepared by mixing (in the order listed) 2.5 μlH₂O, 2.0 μl 10× reaction buffer (Ambion), 10 μl oligo dT (20 pmol), 1.0μl dNTP mix (10 mM each), 0.5 μl RNAsin® (20 U) (Ambion, Inc., Hialeah,Fla.), and 0.5 μl MMLV reverse transcriptase (50 U) (Ambion Inc.). Thecontents are then mixed by pipetting up and down, and the reactionmixture is incubated at 42° C. for 1 hour. The contents of each tube arecentrifuged prior to amplification.

An amplification mixture is prepared by mixing in the following order toobtain the final concentrations listed for each component: 1× PCR bufferII (10 mM Tris pH 8.3, 50 mM KCl), 3 mM MgCl₂, 140 μM each dNTP, 0.175pmol each oligo, 1:50,000 dilution of SYBR® Green, 0.25 mg/ml BSA, 1unit Taq polymerase, and H₂O to 20 μl. To each 20 μl aliquot ofamplification mixture, 2 μl of template RT is added, and amplificationis carried out according to standard protocols. As double stranded PCRproduct is produced during amplification, the fluorescence from SYBR®Green increases.

Example 4 Effect of NRG4 Expression on Proliferation

The effect of NRG4 on proliferation is assessed using antisenseoligonucleotides in metastatic breast cancer cell lines (MDA-MB-231(“231”)), SW620 or Km12L4 colon colorectal carcinoma cells, or 847 humanimmortal fibroblast cells. Transfection is carried out as describedabove in Example 3.

Cells are plated to approximately 50-80% confluence in 96-well dishes.Antisense or reverse control oligonucleotide is diluted to 2 μM inOptiMEM™ and added to OptiMEM™ into which the delivery vehicle, lipitoid116-6 in the case of SW620 cells or 1:1 lipitoid 1:cholesteriod 1 in thecase of MDA-MB-231 cells, is diluted. The oligo/delivery vehicle mixtureis then further diluted into medium with serum on the cells. The finalconcentration of oligonucleotide for all experiments is 300 nM, and thefinal ratio of oligo to delivery vehicle for all experiments is 1.5 nmollipitoid/μg oligonucleotide. Cells are transfected overnight at 37° C.and the transfection mixture is replaced with fresh medium the nextmorning. The cells are counted at various time points, and cellproliferation is determined using CyQuant (Molecular Probes) accordingto the manufacturer's protocol.

Example 5 Effect of NRG4 Expression on Colony Formation

The effect of NRG4 expression on colony formation is tested in a softagar assay. Soft agar assays are conducted by first establishing abottom layer of 2 ml of 0.6% agar in media plated fresh in each well ofa 6-well plate within a few hours of layering on the cells. The celllayer is formed on top of the bottom layer by first obtaining cellstransfected as described above from plates using 0.05% trypsin. Thecells are washed twice in media, counted in a Coulter counter, andre-suspended to 10⁶ per ml in media. 10 μl aliquots are placed withmedia in 96-well plates (to check viable cell count with WST1), ordiluted further for soft agar assay. 2000 cells are plated in 800 μl0.4% agar in duplicate wells above 0.6% agar bottom layer. After thecell layer agar solidifies, 2 ml of media is dribbled on top andantisense or reverse control oligo is added without delivery vehicles.Fresh media and oligos are added every 34 days. Colonies are formed in10 days to 3 weeks. Fields of colonies are counted by eye. Wst-1metabolism values can be used to compensate for small differences instarting cell number. Larger fields can be scanned for visual record ofdifferences.

All publications and patent documents mentioned in the specification areherein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. A method of diagnosing pancreatic cancer in a patient, said methodcomprising comparing the amount of NRG4 expression product in abiological sample acquired from said patient to the amount of NRG4expression product from a normal control, wherein said NRG4 expressionproduct is: (a) a mRNA encoding an NRG4 polypeptide comprising an aminoacid sequence at least 98% identical to SEQ ID NO:2; or (b) a NRG4polypeptide comprising an amino acid sequence at least 98% identical toSEQ ID NO:2, or an immunologically active fragment thereof; whereindecreased amounts of the NRG4 expression product in the biologicalsample acquired from the patient compared to the amount of NRG4expression product from the normal control is indicative of the presenceof pancreatic cancer in said patient.
 2. The method of claim 1 whereinthe NRG4 polypeptide comprises an amino acid sequence at least 99%identical to SEQ ID NO:2.
 3. The method of claim 1 wherein the NRG4polypeptide comprises the amino acid sequence of SEQ ID NO:2.
 4. Themethod of claim 1 wherein the NRG4 polypeptide shows a comparableexpression pattern in cancerous pancreatic cells as a polypeptidecomprising the amino acid sequence of SEQ ID NO:2.
 5. The method ofclaim 1 wherein the NRG4 polypeptide comprises amino acids correspondingto residues 4 to 50 of SEQ ID NO:2.
 6. A method of diagnosing pancreaticcancer in a patient, said method comprising: (a) contacting apolynucleotide that binds to mRNA encoding an NRG4 polypeptidecomprising the amino acid sequence of SEQ ID NO:2 with nucleic acids ofa biological sample acquired from said patient under binding conditionssuitable to form a duplex; and (b) comparing the amount of the duplexformed to the amount of duplex formed when the polynucleotide iscontacted with nucleic acids of a biological sample acquired from anormal control; wherein decreased levels of the amount of duplex formedupon contacting said polynucleotide and said nucleic acids of thebiological sample acquired from said patient compared to the amount ofduplex formed upon contacting said polynucleotide and said nucleic acidsof the biological sample from the normal control is indicative of thepresence of pancreatic cancer in said patient.
 7. The method of claim 6,wherein said polynucleotide is detectably labeled.
 8. The method ofclaim 7, wherein the label is a radioactive, fluorescent, biotinylated,or chemiluminescent label.
 9. A method of diagnosing pancreatic cancerin a patient, said method comprising comparing the amount of mRNAencoding an NRG4 polypeptide comprising an amino acid sequence at least98% identical to SEQ ID NO:2 in a sample acquired from said patient tothe amount of mRNA encoding an NRG4 polypeptide comprising an amino acidsequence at least 98% identical to SEQ ID NO:2 from a normal sample;wherein decreased levels of the amount of mRNA encoding an NRG4polypeptide comprising an amino acid sequence at least 98% identical toSEQ ID NO:2 in the biological sample acquired from said patient comparedto the amount of mRNA encoding an NRG4 polypeptide comprising an aminoacid sequence at least 98% identical to SEQ ID NO:2 acquired from thenormal control is indicative of the presence of pancreatic cancer insaid patient.
 10. The method of claim 9 wherein amounts of mRNA arecompared using Northern blot analysis, primer extension, S1 nucleaseprotection, reverse transcription-polymerase chain reaction (RT-PCR), orin situ hybridization.
 11. The method of claim 9 wherein the NRG4polypeptide comprises an amino acid sequence at least 99% identical toSEQ ID NO:2.
 12. The method of claim 9 wherein the NRG4 polypeptidecomprises the amino acid sequence of SEQ ID NO:2.
 13. The method ofclaim 9 wherein the NRG4 polypeptide shows a comparable expressionpattern in cancerous pancreatic cells as a polypeptide comprising theamino acid sequence of SEQ ID NO:2.
 14. The method of claim 9 whereinthe NRG4 polypeptide comprises amino acids corresponding to residues 4to 50 of SEQ ID NO:2.
 15. A method of diagnosing pancreatic cancer in apatient, the method comprising: (a) providing an antibody or antigenbinding fragment thereof that binds to an NRG4 polypeptide comprising anamino acid sequence at least 98% identical to SEQ ID NO:2 or animmunologically active fragment thereof; (b) contacting said antibody orantigen binding fragment thereof with polypeptides of a biologicalsample acquired from said patient under binding conditions suitable toform a complex; and (c) comparing the amount of the complex formed tothe amount of complex formed when said antibody or antigen bindingfragment thereof is contacted with polypeptides of a biological sampleacquired from a normal control, wherein decreased levels of the amountof complex formed upon contacting said antibody or antigen bindingfragment thereof and said polypeptides of the biological sample acquiredfrom said patient compared to the amount of complex formed uponcontacting said antibody or antigen binding fragment thereof and saidpolypeptides of the biological sample from the normal control isindicative of the presence of pancreatic cancer in said patient.
 16. Themethod of claim 15 wherein the polypeptide or immunological fragmentthereof comprises at least 20 contiguous amino acids from SEQ ID NO:2.17. The method of claim 15 wherein the polypeptide or immunologicalfragment thereof comprises at least 50 contiguous amino acids from SEQID NO:2.
 18. The method of claim 15, wherein said antibody or antigenbinding fragment thereof is detectably labeled with an enzyme,radioisotope or fluorophore.
 19. The method of claim 15, wherein saidantibody or antigen binding fragment thereof is conjugated to acytotoxic agent selected from the group consisting of a toxin, achemotherapeutic agent, an anti-mitotic agent, and an antibiotic. 20.The method of claim 15 wherein the antibody or antigen binding fragmentthereof is selected from the group consisting of a polyclonal antibody,a monoclonal antibody, a chimeric antibody, a humanized antibody, asingle chain antibody, a Fab fragment, and a F(ab′)₂ fragment.
 21. Themethod of claim 15 wherein the polypeptide shows a comparable expressionpattern in cancerous pancreatic cells as a polypeptide comprising theamino acid sequence of SEQ ID NO:2.
 22. The method of claim 15 whereinthe polypeptide comprises an amino acid sequence at least 99% identicalto SEQ ID NO:2, or an immunologically active fragment thereof.
 23. Themethod of claim 15 wherein the polypeptide comprises amino acidscorresponding to residues 4 to 50 of SEQ ID NO:2.